An inhibitor is described that prevents adhesion of specific crystals to the surface of kidney cells and is used in an assay system to rapidly measure relative amounts of crystal adhesion to cells. Uses of the inhibitor include preventing kidney stone disease, identifying individuals at high risk of kidney disease, and screening for drugs which prevent adhesion of crystals to cells.
Kidney diseases are major public health problems. At least 3,000,000 people in the United States are affected annually. A type of kidney disease is the formation of xe2x80x9cstones,xe2x80x9d a process called nephrolithiasis. An estimated 1% of adult males in industrialized countries have xe2x80x9cstones.xe2x80x9d
Although nephrolithiasis is a common disease, the mechanisms by which stones develop in the kidney are poorly understood. Renal tubular fluid is normally supersaturated with calcium and oxalate ions which can nucleate to form crystals of calcium oxalate monohydrate (COM). However, this fact alone does not explain how these crystals are retained in the nephrons of the kidney and produce stones. Moreover, some doubt that crystal formation, per se, results in stones, in part because calculations based on the rate of crystal growth and flow of tubular fluid suggest that a nascent crystal could not become large enough to occlude a tubule lumen during the time required for transit through the nephron. To resolve this problem and explain how stones form, there is speculation that either several small crystals aggregate to form a mass large enough to block a tubule, or small crystals bind to the tubular epithelial cell surface where they accumulate. Otherwise crystals would leave the nephron suspended in the flowing tubular fluid, and kidney stones would not develop from crystals.
Urinary COM crystals are implicated in kidney stone disease, and several different lines of investigation emphasize the importance of crystal-cell interactions in the pathogenesis of nephrolithiasis. Associations between crystals in tubular fluid and renal epithelial cells appear to take place in vivo. Papillary casts are often found in kidney stones, and Randallis plaques are known to form in the renal papillae during crystalluria. Recent investigations show that COM crystals, the most abundant constituent of kidney stones, can rapidly adhere to the surface of kidney tubular cells, undergo internalization, and stimulate gene expression, cytoskeletal reorganization and mitogenesis.
Information on the responses of kidney tubular epithelial cells to COM crystals was provided by observation of humans with hyperoxaluria. Hyperoxaluria can be classified as either primary or secondary and is often associated with interstitial fibrosis and renal failure. Primary hyperoxaluria is a genetically distinct inborn error of oxalate metabolism, whereas secondary hyperoxaluria occurs in several gastrointestinal malabsorptive states, during pyridoxine deficiency, and following ethylene glycol ingestion and methoxyflurane anesthesia. Intracellular calcium oxalate crystals and proliferating tubular cells were noted in human tissue biopsied from a normal kidney 16 days after it was transplanted into a patient with primary hyperoxaluria, and engulfment of calcium oxalate crystals and tubular cell proliferation were also reported in a patient with hyperoxaluria and acute renal failure associated with Crohn""s disease. Crystals were observed within tubular epithelial cells and were associated with proliferation and the formation of multinucleated giant cells. Adhesion of crystals to the apical surface of tubular cells was noted by scanning electron microscopy performed on renal tissue from a patient with hyperoxaluria. The importance of the plasma membrane in crystal-cell interactions was also suggested by the observation that membrane fragments of renal epithelial cells promote crystallization from supersaturated calcium oxalate solutions.
Calcium oxalate crystals, when deposited in the interstitium, can cause marked inflammation and fibrosis of the renal parenchyma. An autopsy study. of persons with normal kidney function, acute renal failure or chronic renal failure revealed that the incidence and severity of tubular and interstitial calcium oxalate deposition was a function of the duration of renal failure which in turn is correlated with an elevated plasma oxalate concentration. Therefore calcium oxalate deposits in the kidney are associated with both interstitial fibrosis and loss of renal function. Crystal endocytosis might occur to a lesser extent in the kidney of normal individuals than in those who form stones.
In an animal model, severe hyperoxaluria induced in rats by an intraperitoneal injection of sodium oxalate immediately produces intraluminal calcium oxalate crystals which attach to the apical membrane of renal tubular epithelial cells and subsequently appear as deposits in the interstitium of the kidney. One possible scenario based on current experimental evidence is that during periods of hyperoxaluria COM crystals can nucleate and grow within tubules, bind to tubular cells, undergo endocytosis, and initiate release of factors from tubular cells that could stimulate fibroblast proliferation by a paracrine pathway and ECM accumulation via the plasmin system. The end result of this pathway is interstitial fibrosis and progressive kidney failure.
The in vitro interaction between kidney cells and crystals was elucidated by utilizing a model system of high-density, quiescent cultures of nontransformed monkey renal epithelial cells (BSC-1 line) to simulate the tubular epithelium. These cultures are prepared by allowing cells to completely cover the surface of a culture dish and then reducing their growth to a minimal level by lowering the concentration of serum to 0.01% in the medium. Exogenous COM crystals irreversibly bound to the cells in culture within as little as 15 seconds, were subsequently endocytosed, and often stimulated proliferation. BSC-1 cells appear to survive and divide in culture despite the presence of internalized COM crystals, providing evidence that the crystals are not toxic for these renal cells. COM crystals are more avidly internalized by BSC-1 cells than two other calcium-containing crystals, hydroxyapatite (HA) or brushite (BR). Given the different molecular structures of crystalline surfaces, it is possible that the affinity of COM crystals for the cell surface is greater than it is for HA or BR crystals. In fact, COM crystals are mitogenic for cultured renal epithelial cells of the BSC-1 and MDCK lines, a unique property not shared by another calcium-containing crystal (brushite) or by latex beads. The uptake of COM crystals by BSC-1 cells is a regulated event which can be modified by diverse signals. The mitogens epidermal growth factor (EGF), adenosine diphosphate (ADP) and calf serum each increase COM crystal endocytosis, whereas urinary Tamm-Horsfall glycoprotein (THP), heparin, transforming growth factor TGF-xcex22, and the tetrapeptide arginine-glycine-aspartate-serine (SEQ ID NO:1) (RGDS) inhibit it. Thus renal epithelial cells respond in a specific pattern to a crystal commonly found in urine, and these responses can be modified by extracellular signals. The appearance of crystals in vivo is similar to those of BSC-1 cells in culture, a nontransformed renal epithelial cell line derived from the African green monkey, suggesting that BSC-1 cells in vitro are a model for renal tubular cell interactions with COM crystals in vivo.
When used as an in vitro system to study the renal cell-crystal interaction, COM crystals were observed to adhere to BSC-1 cells after as little as 15 seconds, far less than the estimated 3-5 minutes required for filtrate to traverse the length of the nephron. Crystals might have prolonged contact with the kidney tubular lining cells in vivo if fluid travels within the tubule in a laminar fashion, as has been proposed, and the flow rate adjacent to the epithelial cell surface approaches zero.
When the most common type of crystal in kidney stones, COM, was added to cultures of monkey kidney epithelial cells (BSC-1 line) 19% of the cells internalized a crystal after 30 minutes.
Endocytosis of COM crystals by cultured renal epithelial cells is regulated by diverse molecules, suggesting that in vivo along the nephron, the outcome of crystal-cell interactions could be determined by the balance between positive EGF, ADP and negative factors (fibronectin, TGF-xcex22) such as those discussed herein. Endocytosis may be mediated in part by a specific cell surface receptor.
Nontransformed monkey renal epithelial cells (BSC-1 line) do not perceive crystals as inert, but respond by displaying a program of specific events including binding of the crystal to the cell surface, crystal endocytosis, proto-oncogene expression, reorganization of actin filaments and cytokeratine-containing intermediate filaments, DNA synthesis, and, in some instances, cell multiplication. The response of renal epithelial cells to COM crystals is characterized by increased expression of specific genes which encode transcriptional activators (c-myc, EGR-1, Nur-77, and c-jun), a regulator of the extracellular matrix (ECM) composition (PAI-1), and growth factors (platelet-derived growth factor [PDGF]-A chain and connective tissue growth factor [CTGF]). The protein products of these genes (PAI-1, PDGF-A chain, CTGF) could contribute to interstitial fibrosis observed in kidneys of patients with primary or secondary hyperoxaluria.
The presence of cell-surface binding sites for COM crystals was suggested by investigations utilizing primary cultures of rat medullary cells. Binding was a saturable process that was partially antagonized by HA crystals. Further study revealed that cells which avidly bound crystals expressed basolateral surface antigens on their apical surfaces. Additional support for specific plasma membrane crystal-binding sites was obtained by treating cells with EGTA which exposed basolateral epitopes and permitted increased COM crystal binding. These experiments, and the observation that COM crystals bind to injured regions of rat bladder epithelium suggest that crystal-binding sites may be minimally exposed under physiological circumstances, but are unmasked when cells are injured, or possibly during regeneration after injury. Specific soluble factors may also modify crystal-cell interactions. In a study of crystal-induced lysis of red blood cells, known inhibitors of crystal growth such as citrate and pyrophosphate were shown to decrease attachment of COM, HA, and monosodium urate crystals to the plasma membrane. The response of a renal epithelial cell to a urinary crystal may not be determined solely by the interaction at the plasma membrane, but may be modulated by biological signals.
The interaction of COM crystals with kidney cells in culture can result in specific responses such as binding to the apical cell surface, internalization, and in some cells initiation of proliferation. Each of these three responses appears to be under the control of a different set of extracellular factors. Crystal binding to the apical plasma membrane can be blocked by diverse anions found in urine such as the glycoproteins nephrocalcin and uropontin, specific glycosaminoglycans, and citrate. After crystals adhere they can be internalized by the cells, a process which can be stimulated (by e.g., EGF, ADP, calf serum)., or inhibited (by e.g., THP, heparin, TGF-xcex22, RGDS). The capacity of THP, fibronectin or heparin to inhibit endocytosis was mediated by an interaction of these molecules with cells, not by coating the crystals. Thus renal epithelial cell endocytosis of COM crystals is regulated by diverse molecules including THP, the most common protein found in human urine. Uptake of COM crystals is associated with an increased probability of cell division, and the internalized crystals can apparently be distributed to daughter cells at mitosis. In addition, the crystals can persist for at least two weeks within the cells suggesting that they are not perceived as toxic. The cell-crystal interaction can stimulate expression of specific genes whose products may contribute to some of these processes, such as cell growth and accumulation of ECM constituents.
Specific responses to crystals also occur in nonrenal cells. Basic calcium phosphate crystals induce c-fos and c-myc protooncogene expression and initiate mitogenesis in Balb/3T3 fibroblasts. A role for cytokines in cell-crystal interactions has also been reported. Monosodium urate, calcium pyrophosphate dihydrate, and hydroxyapatite crystals each stimulated interleukin (IL)-6 production by synoviocytes and monocytes grown in culture, and monosodium urate crystals trigger release of IL-8 from cultured monocytes.
The plasminogen-activating system plays a key role in regulating the extracellular matrix (ECM) composition. Because progressive accumulation of extracellular proteins is a central feature of interstitial fibrosis, genes which regulate the components of the plasminogen-activating system were studied in renal epithelial cells of the BSC-1 line exposed to COM crystals. Plasmin is an extracellular broadspectrum protease that is activated when its precursor, plasminogen, is cleaved. Plasminogen is the target of two other highly specific proteases, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). u-PA is primarily responsible for plasmin generation in processes involving degradation of ECM and basement membranes. Fast-acting plasminogen activator inhibitor (PAI-1) regulates plasmin activity by blocking the action of UPA which decreases formation of plasmin. Reduced plasmin production could thereby permit accumulation of ECM proteins. When Northern analysis was used to study gene expression, PAI-1 was induced and u-PA was unchanged in renal cells exposed to COM crystals. Increased expression of PAI-1 without a change in u-PA could result in decreased plasmin production and enhanced accumulation of ECM proteins so that eventual fibrosis is the predicted result. Augmented expression of the gene encoding PDGF-A chain was also detected. Increased availability of PDGF in the extracellular space would favor fibrosis. CTGF is a peptide originally identified as a secreted product of human vascular endothelial cells that has properties similar to PDGF; it is mitogenic and chemotactic for connective tissue cells such as fibroblasts and smooth muscle cells. Induction of the transcript for CTGF at one hour after exposure to crystals and its persistent expression for the next twenty three hours suggests that secreted CTGF protein could stimulate fibroblast proliferation in a paracrine manner, as does PDGF. Of 15 genes studied which regulate ECM composition, only three (PAI-1, PDGF-A chain, CTGF) exhibited increased expression after exposure of the cells to COM crystal. These results suggest that stimulated gene expression in this setting is highly targeted within the genome.
Early structural and functional changes at the kidney epithelial cell surface were identified during an interaction between a COM crystal and cultured BSC-1 cells. [14C] COM crystals bind to the cell surfaces within seconds. Scanning electron microscopy (SEM) was used to examine the structural correlates of COM crystal binding to the apical membrane of BSC-1 cells. Under low power, the outline of individual cells, nuclei and surface microvilli were seen, as well as small adherent crystal aggregates. Higher magnification revealed contact between microvilli and the crystal surface. At the base of the same microvillus small cellular.extensions could be seen over the surface of the crystal. In other instances extended microvilli covered a substantial portion of the crystal. These microvillar processes appeared to subsequently coalesce and completely cover the crystal. At later times apparent crystal aggregates were observed immediately beneath the plasma membrane. Microvilli on the surface of macrophages appear to contribute to phagocytosis in a similar manner.
Transmission electron microscopy (TEM) was used to visualize intracellular changes as COM crystals were engulfed. Crystals adherent to microvilli were noted. Microvillar processes appeared to extend sequentially to occupy a sizable portion of the crystal surface. Inside cells crystals appeared within membrane-lined vacuoles. Lysosomes were located in the vicinity of intracellular crystals at 3 hours and after 12 hours small crystals were seen within the organelle.
Cytoskeletal responses to crystal uptake were sought by immunofluorescence microscopy which revealed concentration of F-actin at sites of crystal contact as well as a generalized reorganization of the intermediate filament network containing cytokeratin 8.
COM crystals are mitogenic for BSC-1 cells. The fate of crystals in cells going through mitosis was elucidated as follows: Subconfluent cultures of BSC-1 cells were prepared and crystals (50 xcexcg/ml) were added on day zero. On day 1 the medium was changed to remove any nonadherent crystals. The number of cells in the culture containing one or more crystals increased between 1 and 7 days (P less than 0.001), although no additional crystals were added after day 0, demonstrating that internalized crystals were passed on to daughter cells during division. Furthermore, the presence of intracellular crystals did not adversely affect cell growth.
Four signals were identified which alter cellular function, are mitogenic for BSC-1 cells and stimulate endocytosis of COM crystals, although the pathways by which they do so likely differ. The concentrations of EGF and ADP that enhance endocytosis are much below those that initiate DNA synthesis. Two of the regulatory signals identified modify cell structure. Exposure of BSC-1 cells to ADP for 2 minutes induces marked changes in cell shape and reorganization of the intermediate filaments containing cytokeratin 8. A low-potassium (K) environment initiates functional changes in the plasma membrane within seconds, and an increased number of surface microvilli within 3 minutes. Thus when cells are exposed to ADP or low-K medium the cytoskeleton appears to play a role in generating the observed structural changes, and might also mediate crystal uptake.
Factors that regulate endocytosis of COM crystals by kidney epithelial cells are important to identify because increased understanding of mechanisms which mediate formation of a renal calculus can lead to diagnostic assays and treatment for this disease, for example, by use of crystal adhesion inhibitors.
Preventing crystal adhesion to the cell surface is a means to block the cascade of events that results in crystal retention and nephrolithiasis. As illustrated in FIG. 5, by scanning (SEM) and transmission electron microscopy (TEM), COM crystals are seen to first make contact with microvilli on the apical surface of BSC-1 cells. Subsequently, crystals are visualized within membrane lined vesicles, which in some instances fuse with lysosomes. These observations suggest an affinity between the crystals and the kidney cell surface that provokes a cellular response. (Lieske et al., 1994).
A composition which disrupts the cascade of events that results in crystal retention and nephrolithiasis is the crystal adhesion inhibitor (CAI) of the present invention, a composition which is a sialic acid-containing anionic glycoprotein having an estimated molecular weight of 39,000 daltons based on SDS polyacrylamide gel electrophoresis. The sialic acid residues are important in maintaining a negative charge, because the amino acid composition of the inhibitor predicts a net near-neutral charge.
Renal epithelial cells constitutively produce the factor (CAI) that prevents adhesion of COM crystals to their apical surface. Sequence information for 124 amino acids of the NH2-terminus and 6 internal fragments of this novel core protein has been obtained, and used to prepare 2 monospecific antibodies against 2 different synthetic peptides.
The inhibitor is purified by a novel crystal-affinity method wherein the anionic, hydrophobic material adheres to the crystals from which it is later removed by, e.g., EDTA. This is followed by purification using SDS-PAGE and electroblotting or electroelution of the gel or reversed-phase HPLC. The purified CAI is an anionic glycoprotein. The presence of carbohydrate is manifested by a loss of inhibitory activity following exposure of CAI to neuraminidase, indicated the critical functional importance of its anionic sialic acid residues. Its carbohydrate character is confirmed by the detection of uronic acid using the carbazole reaction, and a positive test using a DIG glycan kit. Its protein character is established by amino acid compositional analysis and amino acid sequence information, and is supported by positive reactions in the presence of ninhydrin or bicinchoninic acid (BCA). Its activity is resistant to pH 2, freezing and thawing. The near neutral net charge of the CAI protein distinguishes it from known strongly anionic proteins that block adhesion of COM crystals to the surface of renal epithelial cells.
Availability of the CAI permits comparative screening for other candidate inhibitors of crystal adhesion. Generally, those at least as active in preventing crystal adhesion to cells, are selected for further processing. The tissue culture system of the present invention is contacted with a candidate agent, and the degree of inhibition of crystal adhesion to cells in a control culture are each compared to a culture treated with CAI as a standard.
Assays based on crystal adhesion are useful for identifying patients at high risk for kidney stone disease and for screening for drugs which prevent crystal adhesion. Polyclonal antibodies developed against the CAI by standard methods are used to quantitate the amount of CAI in a sample of urine from an individual with untreated or treated nephrolithiasis, or who is suspected of having this condition.
For characterization of the CAI, a monospecific polyclonal antiserum is preferable to monoclonal antibodies because the latter each complex with single antigenic determinants, whereas a polyclonal antiserum likely recognizes multiple sites on the target molecule. Because CAI is a glycoprotein and neuraminidase treatment inhibits its function, it is likely that sialic acid residues are present at its xe2x80x9cactive site(s)xe2x80x9d, the sites by which the CAI binds to crystal surfaces or cell surfaces to block crystal adhesion. Thus a polyclonal antiserum which contains IgG molecules that recognize antigenic determinants composed of carbohydrate, protein, or both is a particularly useful reagent. Whereas a monoclonal antibody might recognize antigenic sites on the CAI molecule which are not important for its biologic function, a polyclonal antibody is more likely to block activity when it is recognized and binds to CAI. Monospecific antibodies have been prepared to two synthetic peptides having sequences identified in CAI. Monoclonal antibodies can also be prepared to functionally active fragments of the CAI used as immunogens employing techniques well-known to the art.
To determine the minimum amount of the molecule necessary to elicit biological activity, that is, the minimum peptide that includes the xe2x80x9cactive site or sites,xe2x80x9d CAI that has been isolated and purified as described herein is subjected to enzymatic cleavage which produces fragments. The fragments are then tested for biological activity according to the methods described herein. It is likely that the active site or sites will include sialic acid residues. The relative efficiency of the active-site containing fragments is also of interest because even though biologically active, some fragments are likely to be more active than others, e.g. a heparin molecule of 5,000 daltons molecular weight is not as efficient as a heparin molecule of 18,000 daltons molecular weight. Inhibitors are designed to complex with the active site(s) as determined by the methods discussed above.
Susceptibility to stone formation varies among individuals. This variation is likely due to inherent variations among individuals in the ability to produce inhibitor, which is measurable and provides a means for classifying persons according to risk of developing stones. If an immunological assay detects no CAI or an amount of CAI less than the value in nonaffected control individuals, the patient is considered to be at increased risk of kidney stone formation. This assay is also useful for monitoring the success of therapeutic regimens designed to treat or prevent the appearance of new stones which may be directly correlated to the urinary concentration of CAI in specific individuals. The amount of CAI detected by the immunological assay and its functional capacity to inhibit adhesion of COM crystals to kidney epithelial cells in culture is used to classify patients with nephrolithiasis. A tissue culture system containing kidney epithelial cells is used to quantitate the function of the CAI or its equivalent.
Novel molecular tools are now available to seek the CDNA sequence of CAI. Amino acid sequence information now available (124 residues) permits studies using a polymerase chain reaction (PCR) strategy, whereas the two monospecific antisera are useful in an immunoscreening approach to obtain a cDNA sequence.
After the DNA sequence of the gene encoding CAI is obtained, further study of the protein and the factors that regulate its production will improve understanding of kidney stone formation. In certain individuals, defective production of CAI likely predisposes to kidney stone formation. In CAI-deficient patients, adhesion of crystals to tubular cells is expected to occur more readily, and once retained in the nephron, such crystals likely grow into kidney stones. Therefore, study of CAI could yield important new insights into the pathogenesis of kidney stone formation, and lead to development of a novel form of therapy by using knowledge about CAI structure and function.
To summarize uses of CAI:
1. Urine from a patient is tested to detect defective CAI or other anti-adhesion factors in urine by using the crystal adhesion assay described herein.
2. Diverse chemical and pharmaceutical agents are selected because they exhibit characteristics similar to CAI, and are tested for their ability to prevent crystal adhesion to cells as compared to CAI.
3. The structure of CAI provides a basis for the rational design of effective drugs, for example, an active domain of the CAI molecule that is smaller than the native molecule in size may be used to prevent crystal adhesion.
4. CAI is used as a drug to prevent stones: delivery is via liposome, intravenous or subcutaneous injection, or intranasal systems. Chemical modification of CAI may permit its use by an oral route.
5. Monospecific antibodies to purified CAI or peptide fragments of CAI are provided for detection and quantitative assays of CAI.
6. Hybridization of CAI cDNA with samples of DNA from individuals is used to screen for defective CAI carriers who are candidates for stone disease. Sloughed renal tubular cells isolated from urine or white blood cells from peripheral blood are suitable samples for this hybridization test.
7. If a crystal-binding receptor is identified on a cell surface, sloughed renal tubular cells could be screened for an abnormal quantity/quality of the receptor.